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Ischemia and hypoxia cause functional damage to brain tissues during stroke， and when blood supply is restored to brain tissues after ischemia， a large number of free radicals and calcium overload cause cerebral ischemia-reperfusion injury， which further aggravates the condition. Autophagy is a self-protection mechanism that maintains the homeostasis of the intracellular environment， but excessive autophagy causes brain tissue damage. MiRNA is a small endogenous non-coding RNA molecule that regulate various physiological activities at the gene level by binding to complementary sequences in the 3 '- UTR of its target gene mRNA， leading to translation inhibition or mRNA degradation. MiRNA not only directly acts on autophagy related proteins， but also participates in autophagy regulation induced by ischemia/reperfusion through various signaling pathways. However， there is still a lack of systematic induction and analysis of miRNA regulation of autophagy signaling pathways induced by cerebral ischemia/reperfusion. This article reviews the regulation of cellular autophagy during cerebral ischemia/ reperfusion by miRNA-124， miRNA-298， miRNA-202-5p， miRNA-142， miRNA-26b and so on through different signaling pathways， providing a systematic and theoretical approach for the study of autophagy in stroke.
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Cerebral ischemia/reperfusion injury （CIRI） is a complex cascade reaction process in which the blood flow and oxygen supply of brain tissue in the infarcted area recover after cerebral ischemia， resulting in secondary injury of ischemic brain tissue. At present， thrombolysis as soon as possible and restoration of cerebral blood supply are still the only strategies for the treatment of stroke， but a considerable number of patients' symptoms will be more serious after reperfusion， making patients face adverse outcomes such as neurological function injury and even death and seriously affecting the quality of life and safety of patients. Therefore， an in-depth exploration of the mechanism and treatment strategy of CIRI has important clinical significance. The phosphatidylinositol 3- kinase （PI3K）/protein kinase B （Akt） signaling pathway is one of the classic anti-apoptosis/reproductive-promoting signal transduction pathways， which is responsible for cell proliferation， growth， and differentiation. It is the key cascade signaling pathway of CIRI， located at the core site in many mechanisms such as mitochondrial disorder， apoptosis， autophagy， oxidative stress， and inflammation. It is closely related to the occurrence and development of CIRI. Traditional Chinese medicine has been used in the clinical treatment of stroke and its complications for thousands of years， and the clinical effect of traditional Chinese medicine in the prevention and treatment of CIRI has been affirmed by a large number of research results in recent years. It is further clarified that the monomers， active components， and their compound prescriptions of traditional Chinese medicine can directly or indirectly regulate the PI3K/Akt signaling pathway by virtue of the biological advantages of multi-targets， multi-components， and multi-pathways and play an overall protective role in CIRI. By analyzing the related research progress of traditional Chinese medicine in China and abroad in recent years， the authors summarized the role and mechanism of regulating the PI3K/Akt signaling pathway in the prevention and treatment of CIRI， so as to provide further theoretical basis for the study of the mechanism of clinical prevention and treatment of CIRI.
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Objective To investigate the potential effect and mechanism of curcumin in inhibiting synaptic injury in the cortex of rats with cerebral ischemia-reperfusion. Methods Sprague-Dawley rats were divided into sham-operated group, model group, low-dose curcumin (50 mg/kg) group, and high-dose curcumin (100 mg/kg) group. A model of middle cerebral artery occlusion for 2 hours and reperfusion for 24 hours was constructed, and curcumin was administered. Based on the neurological function score, the effects of curcumin on cerebral infarct volume, synaptic ultrastructure changes, inflammatory cell infiltration, and the expression of NLRP3, Caspase-1, Synapsin1, and CAMKⅡ were observed after the end of the animal treatment. Results The neurological function scores were 0, 3.25±0.43, 2.50±0.50, and 1.50±0.50 for the sham-operated group, model group, low-dose curcumin group, and high-dose curcumin group, respectively. The percentage of cerebral infarct volume was 0, (38.89±2.21)%, (33.48±1.77)%, and (23.69±2.19)%, respectively. Compared with the sham operation group, the model group had severe synaptic ultrastructure damage, extensive inflammatory cell infiltration, significantly increased expression of Caspase-1 and NLRP3 (P < 0.5), and significantly decreased expression of Synapsin1 and CAMKⅡ (P < 0.5). Curcumin treatment significantly inhibited synaptic damage, reduced inflammatory cell infiltration, decreased the expression of Caspase-1 and NLRP3 (P < 0.5), and increased the expression of Synapsin1 and CAMKII (P < 0.5), when compared with the model group. Conclusion Ischemia-reperfusion-mediated synaptic injury in rat brain triggers an inflammatory response in cortical nerve cells, and curcumin alleviates synaptic damage and reduces brain injury by inhibiting inflammatory factor levels.
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AIM: To investigate the effect of gastrodin on the expression of brain-derived neurotrophic factor (BDNF) and interleukin-6 (IL-6) in the striatum of cerebral ischemia rats, and to explore the potential mechanism of gastrodin in treating cerebral ischemia. METHODS: The rats were randomly divided into four groups: normal, sham, model, and gastrodin groups, each consisting of 10 rats. After successful modeling using middle cerebral artery occlusion (MCAO), the gastrodin group received intraperitoneal injection of gastrodin injection at a dose of 10 mg/kg once a day for 14 consecutive days. Pathological changes in striatal neurons were observed using Nissl staining. Immunohistochemistry was utilized to detect positive expression of BDNF and IL-6 proteins in the striatum. Additionally, immunoblot analysis was performed to determine the expression levels of BDNF and IL-6 proteins in the striatum. RESULTS: Nissl staining revealed clear and intact structures of striatal neurons in the normal and sham groups, with tightly arranged cells. In the model group, the number of cells was significantly reduced compared to the sham group (P0.05). Compared to the sham group, the model group showed a decrease in the protein expression level of BDNF in the striatum on the ischemic side (P<0.01) and an increase in the protein expression level of IL-6 (P<0.05, P<0.01). In contrast, the gastrodin group showed an increase in the protein expression level of BDNF in the striatum on the ischemic side (P<0.05, P<0.01) and a decrease in the protein expression level of IL-6 (P< 0.05, P<0.01) compared to the model group. CONCLUSION: Gastrodin has a significant protective effect on striatal injury caused by cerebral ischemia, and its mechanism may be related to the up-regulation of the anti-inflammatory factor BDNF and the down-regulation of the pro-inflammatory factor IL-6.
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Aim To investigate the regulatory effect of geraniol on Nrf2/HO-1 signaling pathway after cerebral ischemia-reperfusion(I/R)in rats. Methods In this experiment,all the male SD rats were randomly divided into nine groups receiving the following treatments:sham operation(sham); sham operation+200 mg·kg-1 geraniol; I/R; I/R+50 mg·kg-1 geraniol; I/R+100 mg/kg geraniol; I/R+200 mg·kg-1 geraniol; edaravone; I/R+ brusatol(Nrf2 inhibitor); I/R+200mg·kg-1 geraniol+brusatol. All rats received intraperitoneal injection of geraniol for five consecutive days before MCAO and again after MCAO. During the construction of cerebral I/R injury models,the blood vessels were isolated without any suture in the sham operation and the sham operation +200 mg·kg-1 geraniol groups while the blood vessels with suture in other groups. The damage of neurological function was evaluated by the modified rating scale for neurological function. The TTC,HE and Tunel staining methods were used to determine the cerebral infarction volume,the damage of the ischemic cortex and the apoptosis of cortical cells,respectively. The oxidative stress-related parameters then were measured. The protein expressions of Nrf2 and HO-1 were detected by immunohistochemical staining and the target protein expressions of the injured cortex were detected by Western blot. Results Compared with the model group,it was found that the geraniol treatment significantly repaired neural injury,reduced cerebral infarction volume,cerebral cortex damage and cell apoptosis. Meanwhile,geraniol intervention could significantly increase the expression of Nrf2/HO-1 protein in the right-sided cortex and reduce oxidative stress level. Conclusion Geraniol can attenuate cerebral injury induced by ischemia-reperfusion in rats via activating Nrf2/HO-1 signaling pathway.
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Aim To investigate the dynamic time-course changes in neuronal cytoskeleton after acute ischemia and reperfusion in rats. Methods Reperfusion was performedin rats by blocking the middle cerebralarteryfor 90 min, then therats wereobserved and collected at different time points. The brain damage wasobserved by Nissl staining,and neurobehavioural function was evaluated with neurological deficit score and forelimb placement test. The cellular changes in the alternations of cytoskeletal elements including microtubule associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) were observed by immunohistochemistry staining and Western blot. Impaired axons, dendrites and cytoskeletal alternations were detected by electron microscope. Results Brain damage and neurobehavioural function were gradually aggravated with the prolongation of reperfusion. Brain damage appeared earlier and more severe in striatum than in cortex. Moreover, decreased MAP2-related and increased NF-H-related immunoreactive intensities were found in the ischemic areas. Impaired cytoskeletal arrangement and reduced dense were indicated. Damaged cytoskeletal components such as microtubules and neurofilament arrangement, decreased axonal filament density, and swelled dendrites were observed after cerebral ischemia reperfusion by ultrastructural observations. Conclusions Different brain regions have diverse tolerance to ischemia-reperfusion injury. Major elements of neuronal cytoskeleton show dynamic responses to ischemia and reperfusion, which may further contribute to brain damage and neurological impairment following MCAO and reperfusion.
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Aim To investigate whether alisol A (AA) could improve the blood brain barrier (BBB) mediated cortex cerebral ischemia-repeifusion injury (CIRI) by inhibiting matrix metalloproteinase 9 (MMP-9). Methods The global cerebral ischemia- reperfusion (GCI/R) model in mice was established, and the AA was intragastric injected subsequently for seven days. The modified neurological severity scores (mNSS), open field test and Y-maze test were applied to detect neurological function. Magnetic resonance spectroscopy (MRS) was used to detect relevant neu- rosubstance metabolism in cortex of mice. Transmission electron microscope (TEM) was employed to observe the ultrastructure of BBB in cortex. Western blot and immunohistochemistry were used to detect the MMP-9 level in cortex. The binding possibility of A A and MMP-9 was determined by molecular docking. Results Compared with Sham group, mice in GCI/R group have an increased mNSS score but decreased at total distance and center distance to total distance ratio in open field test as well as alternation rate in Y-maze test (P<0.01). While mice in GCI/R + AA group have a decreased mNSS score but increased at total distance and center distance to total distance ratio in open field test as well as alternation rate in Y-maze test (P<0.01) compared with GCI/R group. MRS results found that in cortex of GCI/R group mice, the level of GABA and NAA significantly decreased while the Cho, mI and Tau level increased (P<0.01). Whereas in GCI/R + AA group mice, the GABA and NAA level increased and the Cho, ml and Tau decreased significantly (P<0.01). By TEM we observed that the basilemma of cerebral microvessels collapsed, the lumen narrowed, the endothelial cells were active and plasma membranes ruffled, gaps between cells were enlarged and tight junctions were damaged and the end feet of astrocytes were swollen in GCI/R group mice. While in GCI/R + AA group mice, the lumen was filled, plasma membranes of endothelial cells were smooth, tight junctions were complete and end feet of astrocytes were in normal condition. Western blot and immunohistochemistry both found that the MMP-9 level increased in GCI/R group mice (P < 0.01) and decreased in GCI/R + AA group mice (P < 0.05). Molecular docking proved the binding between aliso A and MMP9 through TYR-50 and ARG-106, and the binding energy was calculated as -6.24 kcal · mol
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OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemia- reperfusion injury in rats. METHODS The model of middle cerebral artery occlusion （MCAO） was established by using the improved thread occlusion method. The experiment was divided into six groups： sham surgery group （only separating blood vessels without inserting thread plugs， given the same volume of normal saline）， model group （modeling， given the same volume of normal saline）， nimodipine group （positive control， modeling， dose of 20 mg/kg）， and low-dose， medium-dose， and high-dose groups of Longshengzhi capsules （modeling， doses of 0.72， 1.44 and 2.88 g/kg， respectively）， with 10 mice in each group. Each group was given corresponding medication solution/normal saline by gavage， once a day， for 7 consecutive days. One hour after the last administration， the Zea Longa scoring method was used to score the neurological deficits in each group of rats， and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rats； TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio. Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats. Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats. Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the brain tissue of rats. Western blot assay was adopted to detect the relative expressions of TLR4， NLRP3 and phosphorylated NF-κB （p-NF-κB） protein in the brain tissue of rats and its intracellular NF-κB protein. RESULTS Compared with the sham surgery group， the neural dysfunction score， serum levels of TNF-α and IL-6， cerebral infarction volume ratio， relative expression levels of NF-κB and TLR4 mRNA， as well as protein relative expressions of TLR4， NLRP3 and p-NF-κB in the brain tissue， and relative protein expression of intracellular NF-κB were increased significantly in the model group （P＜0.01）； the enlarged gap and significant edema were observed in cortical nerve cells of brain tissue in rats， with a large amount of inflammatory cell infiltration； the positive expression of NLRP3 protein in brain tissue of rats obviously increased. Compared with the model group， the levels of the above indicators in the medium-dose and high-dose groups of Longshengzhi capsules， as well as the Nimodipine group， were reversed to varying degrees， and most differences were statistically significant （P＜0.05 or P＜0.01）； the pathological morphology observation showed a significant improvement， and the positive expression of NLRP3 protein in the brain tissue of rats was obviously reduced. CONCLUSIONS Longshengzhi capsules may inhibit TLR4/NF-κB/NLRP3 signaling pathway and neuroinflammatory response， thereby achieving a protective effect against cerebral ischemia-reperfusion injury in rats.
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Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.
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Animals , Rats , Reperfusion Injury/prevention & control , Brain Ischemia , Protein Kinase C/metabolism , Protein Kinase C/pharmacology , Ischemic Attack, Transient , Oxidative Stress , Neuroprotective Agents/pharmacology , Dexmedetomidine/pharmacology , Heme Oxygenase-1/metabolism , Heme Oxygenase-1/pharmacology , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/pharmacology , Heme Oxygenase (Decyclizing)/pharmacology , Antioxidants/metabolism , Antioxidants/pharmacologyABSTRACT
ObjectiveAfter the brain and heart injuries were simulated by myocardial injury caused by acute cerebral ischemia， this study explored the mechanism of Naoxintong capsules in treating brain and heart injuries under cerebral ischemia state with Toll-like receptor （TLR） 2/TLR4 as the breakthrough point. MethodC57BL/6 male mice were randomly assigned into the sham operation， model， Naoxintong， and Ginaton groups. The middle cerebral artery occlusion （MCAO） method was used to establish a mouse model of cerebral ischemia. The neuroethological score， cerebral infarction area， cell apoptosis， ionized calcium-binding adaptor molecule 1 （IBA-1）-positive microglia proportion， and serum levels of N-terminal pro-brain natriuretic peptide （NT-proBNP）， creatine kinase-MB （CK-MB）， and lactic dehydrogenase （LDH） were determined to evaluate the pharmacodynamic effects of Naoxintong capsules on heart and brain injuries after cerebral ischemia in mice. Western blotting was employed to determine the expression of TLR2/TLR4 protein in the brain and heart of mice. ResultCompared with the sham operation group， the model group showed increased cerebral infarction area， neuroethological score， apoptosis rate， IBA-1-positive microglia proportion， and serum levels of NT-proBNP， CK-MB， and LDH （P<0.01）. Naoxintong capsules reduced the cerebral infarction area， neuroethological score， apoptosis rate， IBA-1-positive microglia proportion （P<0.01）， and serum NT-proBNP and CK-MB levels （P<0.05） in mice compared with the model group. Western blotting results showed that Naoxintong Capsules down-regulated the expression levels of TLR2 （P<0.05） in the brain and TLR2 （P<0.01） and TLR4 （P<0.05） in the heart. ConclusionCerebral ischemia can cause myocardial damage， reflecting the pathological process of cardiac injury after cerebral ischemia. Naoxintong capsules can mitigate brain and heart injuries after cerebral ischemia and achieve the simultaneous treatment of the brain and the heart， in which TLR2/TLR4 plays a role.
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@#ObjectiveTo explore the protective effect of Sox11 gene on cerebral ischemia reperfusion injury(CIRI)in mice and its mechanism,so as to provide a new target for the treatment of CIRI.MethodsThe mouse middle cerebral artery occlusion(MCAO)model and Neuro2A cell oxygen glucose deprivation reperfusion(OGDR)model were established and detected for the temporal and spatial distribution of Sox11 in the models by real-time quantitative PCR,Western blot,immunohistochemistry(IHC)and immunohistofluorescence(IHF).The altered expression of some crucial genes in the pathway of apoptosis and inflammation in OGDR model after the disruption of Sox11 expression was detected by Western blot.ResultsThe expression level of Sox11 mRNA and protein increased significantly in both MCAO and Neuro2A OGDR models(P = 0.000 1 ～ 0.038 8);After CIRI,Sox11 expression was elevated in the hippocampal dentate gyrus(DG)region of mice;After interfering with the expression of Sox11 in OGDR model,the expression of apoptosis-related protein Cleaved Caspase 3 significantly increased,while the expression of anti-apoptosis protein Bcl-2 significantly decreased,and the expression of phosphorylated NF-κB(p-NF-κB)protein related to inflammatory reaction also up-regulated significantly.Conclusion Sox11gene had a protective effect against CIRI in mice,and was involved in the regulation of apoptosis and inflammation pathways after CIRI.
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ObjectiveTo investigate the effects of dopamine receptor agonist pramipexole and levodopa on emotion and cognition, and mitochondrial membrane potential of rats after global cerebral ischemia-reperfusion injury. MethodsA total of 80 male Sprague-Dawley rats were divided into sham group (n = 20), model group (n = 20), pramipexole group (n = 20) and combined group (n = 20). The latter three groups were used to prepare the model of global cerebral ischemia-reperfusion injury with Pulsinelli's four-vessel occlusion. The pramipexole group was intraperitoneally injected pramipexole 0.5 mg/kg once a day, while the combined group was injected levodopa 50 mg/kg and pramipexole 0.5 mg/kg, for 14 days. Five rats in each group were tested with open field test three, seven and 14 days after modeling; five were tested with Y-maze test seven and 14 days after modeling; five were detected mitochondrial membrane potential three, seven and 14 days after modeling; and five were observed under Nissl's staining14 days after modeling. ResultsCompared with the model group, the number of entries into the central zone (P < 0.05), total distance travelled (P < 0.05) and average velocity (P < 0.05) in the open field test increased in the pramipexole and combined groups seven and 14 days after modeling, duration spent in the central zone increased in the pramipexole and combined groups seven days after modeling (P < 0.05); the rate of spontaneous alternation of Y-maze test increased in the pramipexole and combined groups 14 days after modeling (P < 0.05); mitochondrial membrane potential in hippocampus increased in the pramipexole and combined groups seven and 14 days after modeling (P < 0.05), and it was less in the pramipexole group than in the combined group 14 days after modeling (P < 0.05); and the number of surviving neurons in the hippocampal CA1 increased in the pramipexole and combined groups 14 days after modeling (P < 0.05). ConclusionPramipexole may improve emotion and cognition of rats after global cerebral ischemia-reperfusion injury, and it may be helpful for restoring mitochondrial membrane potential as combining with levodopa.
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OBJECTIVE@#To improve the classical 4-vessel occlusion (4VO) model established by Pulsinelli and Brierley.@*METHODS@#Thirty-two male SD rats were randomized into sham operation group, I4VO-Con10 group, I4VO-Int10 group and I4VO-Int15 group. The sham surgery group underwent exposure of the bilateral vertebral arteries and carotid arteries without occlusion to block blood flow. The I4VO-Con10 group experienced continuous ischemia by occluding the bilateral vertebral arteries and carotid arteries for 10 minutes followed by reperfusion for 24 hours. The I4VO-Int10 and I4VO-Int15 groups were subjected to intermittent ischemia. The I4VO- Int10 group underwent 5 minutes of ischemia, followed by 5 minutes of reperfusion and another 5 minutes of ischemia, and then reperfusion for 24 hours. The I4VO-Int15 group experienced 5 minutes of ischemia followed by two cycles of 5 minutes of reperfusion and 5 minutes of ischemia, and then reperfusion for 24 hours. The regional cerebral blood flow (rCBF) was monitored with laser Doppler scanning, and survival of the rats was observed. HE staining was used to observe hippocampal pathologies to determine the optimal method for modeling. Another 48 rats were randomized into 6 groups, including a sham operation group and 5 model groups established using the optimal method. The 5 I4VO model groups were further divided based on the reperfusion time points (1, 3, 7, 14, and 28 days) into I4VO-D1, I4VO-D3, I4VO-D7, I4VO- D14, and I4VO- D28 groups. Body weight changes and survival of the rats were recorded. HE staining was used to observe morphological changes in the hippocampal, retinal and optic tract tissues. The Y-maze test and light/dark box test were used to evaluate cognitive and visual functions of the rats in I4VO-D28 group.@*RESULTS@#Occlusion for 5 min for 3 times at the interval of 5 min was the optimal method for 4VO modeling. In the latter 48 rats, the body weight was significantly lower than that of the sham-operated rats at 1, 3, 7, 14 and 28 days after modeling without significant difference in survival rate among the groups. The rats with intermittent vessel occlusion exhibited progressive deterioration of hippocampal neuronal injury and neuronal loss. Cognitive impairment was observed in the rats in I4VO-D28 group, but no obvious ischemic injury of the retina or the optic tract was detected.@*CONCLUSION@#The improved 4VO model can successfully mimic the main pathological processes of global cerebral ischemia-reperfusion injury without causing visual impairment in rats.
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Rats , Male , Animals , Rats, Sprague-Dawley , Brain Ischemia , Cerebral Infarction , Reperfusion Injury , Body WeightABSTRACT
OBJECTIVE@#To explore the effect and mechanism of schisandrin B (Sch B) in the treatment of cerebral ischemia in rats.@*METHODS@#The cerebral ischemia models were induced by middle cerebral artery occlusion (MCAO) and reperfusion. Sprague-Dawley rats were divided into 6 groups using a random number table, including sham, MCAO, MCAO+Sch B (50 mg/kg), MCAO+Sch B (100 mg/kg), MCAO+Sch B (100 mg/kg)+LY294002, and MCAO+Sch B (100 mg/kg)+wortmannin groups. The effects of Sch B on pathological indicators, including neurological deficit scores, cerebral infarct volume, and brain edema, were subsequently studied. Tissue apoptosis was identified by terminal transferase-mediated dUTP nick end-labeling (TUNEL) staining. The protein expressions involved in apoptosis, inflammation response and oxidative stress were examined by immunofluorescent staining, biochemical analysis and Western blot analysis, respectively. The effect of Sch B on phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling was also explored.@*RESULTS@#Sch B treatment decreased neurological deficit scores, cerebral water content, and infarct volume in MCAO rats (P<0.05 or P<0.01). Neuronal nuclei and TUNEL staining indicated that Sch B also reduced apoptosis in brain tissues, as well as the Bax/Bcl-2 ratio and caspase-3 expression (P<0.01). Sch B regulated the production of myeloperoxidase, malondialdehyde, nitric oxide and superoxide dismutase, as well as the release of cytokine interleukin (IL)-1 β and IL-18, in MCAO rats (P<0.05 or P<0.01). Sch B promoted the phosphorylation of PI3K and AKT. Blocking the PI3K/AKT signaling pathway with LY294002 or wortmannin reduced the protective effect of Sch B against cerebral ischemia (P<0.05 or P<0.01).@*CONCLUSIONS@#Sch B reduced apoptosis, inflammatory response, and oxidative stress of MCAO rats by modulating the PI3K/AKT pathway. Sch B had a potential for treating cerebral ischemia.
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ObjectiveTo investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway . MethodSixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg-1）］， JNK inhibitor SP600125 （SP） group（5 mg·kg-1）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg-1）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （ig） for 3 consecutive days， and the other groups were given equal volume of normal saline （ig）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham group， the model group had elevated neurological deficit score （P<0.05）， enlarged cerebral infarct volume （P<0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （P<0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （P<0.05）， while down-regulated protein expressions of p62 and Bcl-2 （P<0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （P<0.05） while the mRNA expressions of p62 and Bcl-2 were lower （P<0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （P<0.05）， and the cerebral infarct volume was reduced （P<0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （P<0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（P<0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（P<0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（P<0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（P<0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（P<0.05）.Scores of the Ani group were raised （P<0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （P<0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （P<0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （P<0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（P<0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（P<0.05） and Bcl-2 had a down-regulation in the Ani group（P<0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（P<0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（P<0.05）. ConclusionHuazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.
ABSTRACT
Cerebral ischemia-reperfusion is a serious cerebrovascular disease with high morbidity and mortality. In recent years， reperfusion therapy based on thrombolysis and thrombectomy has been the main treatment method for patients with ischemic stroke. Numerous studies have shown that Chinese medicine saponins can effectively interfere with cerebral ischemia-reperfusion in a multi-target and multi-way manner， which have great potential on the treatment and prevention of cerebral ischemia-reperfusion. Taking China National Knowledge Infrastructure （CNKI） literature database and Wanfang literature database as the analysis sources， this paper used SPSS statistics to summarize the number of papers on the treatment of cerebral ischemia-reperfusion with Chinese medicine saponins and CiteSpace to conduct cluster analysis on the high-frequency keywords of the research， thereby expounding the research hotspots and research status of Chinese medicine saponins in the treatment of cerebral ischemia-reperfusion. Based on literature analysis and summary of animal experiments on the treatment of cerebral ischemia-reperfusion with Chinese medicine saponins in the past two decades， Chinese medicine saponins exerted effects by anti-inflammation， inhibition of oxidative stress， immune regulation， protection of nerve cells， inhibition of thrombosis， promotion of thrombolysis， protection of mitochondria， blood-brain barrier repairing， and other ways. The specific mechanism， therapeutic effect， and signaling pathway of each Chinese medicine saponin have been summarized in this study， which provide a theoretical basis for the in-depth research， new drug development， and clinical application of Chinese medicine saponins for the treatment of cerebral ischemia- reperfusion.
ABSTRACT
This study aims to explore the effect of Xiaoxuming Decoction on synaptic plasticity in rats with acute cerebral ischemia-reperfusion. A rat model of cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion(MCAO). Rats were randomly assigned into a sham group, a MCAO group, and a Xiaoxuming Decoction(60 g·kg~(-1)·d~(-1)) group. The Longa score was rated to assess the neurological function of rats with cerebral ischemia for 1.5 h and reperfusion for 24 h. The 2,3,5-triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were employed to observe the cerebral infarction and the pathological changes of brain tissue after cerebral ischemia, respectively. Transmission electron microscopy was employed to detect the structural changes of neurons and synapses in the ischemic penumbra, and immunofluorescence, Western blot to determine the expression of synaptophysin(SYN), neuronal nuclei(NEUN), and postsynaptic density 95(PSD95) in the ischemic penumbra. The experimental results showed that the modeling increased the Longa score and led to cerebral infarction after 24 h of ischemia-reperfusion. Compared with the model group, Xiaoxuming Decoction intervention significantly decreased the Longa score and reduced the formation of cerebral infarction area. The modeling led to the shrinking and vacuolar changes of nuclei in the brain tissue, disordered cell arrangement, and severe cortical ischemia-reperfusion injury, while the pathological damage in the Xiaoxuming Decoction group was mild. The modeling blurred the synaptic boundaries and broadened the synaptic gap, while such changes were recovered in the Xiaoxuming Decoction group. The modeling decreased the fluorescence intensity of NEUN and SYN, while the intensity in Xiaoxuming Decoction group was significantly higher than that in the model group. The expression of SYN and PSD95 in the ischemic penumbra was down-regulated in the model group, while such down-regulation can be alleviated by Xiaoxuming Decoction. In summary, Xiaoxuming Decoction may improve the synaptic plasticity of ischemic penumbra during acute cerebral ischemia-reperfusion by up-regulating the expression of SYN and PSD95.
Subject(s)
Rats , Animals , Rats, Sprague-Dawley , Brain Ischemia/drug therapy , Reperfusion Injury/metabolism , Infarction, Middle Cerebral Artery , Neuronal Plasticity , ReperfusionABSTRACT
Based on the O-GlcNAc transferase(OGT)-PTEN-induced putative kinase 1(PINK1) pathway, the mechanism of 3,4-dihydroxybenzaldehyde(DBD) on mitochondrial quality control was investigated. Middle cerebral artery occlusion/reperfusion(MCAO/R) rats were established. SD rats were randomized into sham operation group(sham), model group(MCAO/R), DBD-L group(5 mg·kg~(-1)), and DBD-H group(10 mg·kg~(-1)). After 7 days of administration(ig), MCAO/R was induced in rats except the sham group with the suture method. Twenty-four h after reperfusion, the neurological function and the percentage of cerebral infarct area were measured. Based on hematoxylin and eosin(HE) staining and Nissl staining, the pathological damage of cerebral neurons was examined. Then the ultrastructure of mitochondria was observed under the electron microscope, and the co-localization of light chain-3(LC3), sequestosome-1(SQSTM1/P62), and Beclin1 was further detected by immunofluorescence staining. It has been reported that the quality of mitochondria can be ensured by inducing mitochondrial autophagy through the OGT-PINK1 pathway. Therefore, Western blot was employed to detect the expression of OGT, mitophagy-related proteins PINK1 and E3 ubiquitin ligase(Parkin), and mitochondrial kinetic proteins dynamin-like protein 1(Drp1) and optic atrophy 1(Opa1). The results showed that MCAO/R group had neurological dysfunction, large cerebral infarct area(P<0.01), damaged morphological structure of neurons, decreased number of Nissl bodies, mitochondrial swelling, disappearance of mitochondrial cristae, decrease of cells with LC3 and Beclin1, rise of cells with P62(P<0.01), inhibited expression of OGT, PINK1, and Parkin, up-regulated expression of Drp1, and down-regulated expression of Opa1 compared with the sham group(P<0.01). However, DBD improved the behavioral deficits and mitochondrial health of MCAO/R rats, as manifested by the improved morphology and structure of neurons and mitochondria and the increased Nissl bodies. Moreover, DBD increased cells with LC3 and Beclin1 and decreased cells with P62(P<0.01). In addition, DBD promoted the expression of OGT, PINK1, Parkin, and Opa1 and inhibited the expression of Drp1, enhancing mitophagy(P<0.05, P<0.01). In conclusion, DBD can trigger PINK1/Parkin-mediated brain mitophagy through the OGT-PINK1 pathway, which plays a positive role in maintaining the health of the mitochondrial network. This may be a mitochondrial therapeutic mechanism to promote nerve cell survival and improve cerebral ischemia/reperfusion injury.
Subject(s)
Animals , Rats , Rats, Sprague-Dawley , Beclin-1 , Mitochondria , Cerebral Infarction , Protein KinasesABSTRACT
OBJECTIVE@#To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI.@*METHODS@#A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1β and IL-18 in the cerebral cortex of rats were determined by ELISA.@*RESULTS@#Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1β and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1β and IL-18 were higher (P<0.01).@*CONCLUSION@#Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.
Subject(s)
Male , Animals , Rats , Rats, Sprague-Dawley , PPAR gamma/genetics , Pyroptosis , Interleukin-18 , Electroacupuncture , NLR Family, Pyrin Domain-Containing 3 Protein , Cerebral Cortex , Cerebral Infarction/therapy , Caspases , RNA, MessengerABSTRACT
OBJECTIVE@#To investigate the neuroprotective effect of electroacupuncture (EA) at "Quchi" (LI 11) and "Zusanli" (ST 36) in the rats with cerebral ischemic reperfusion and the potential mechanism of microglia pyroptosis.@*METHODS@#Sixty SD rats were randomly divided into a sham-operation group, a model group and an EA group, with 20 rats in each group. The Zea Longa method was employed to establish the rat model of the middle cerebral artery occlusion and reperfusion (MACO/R) in the left brain. In the EA group, since the 2nd day of modeling, EA was given at "Quchi" (LI 11) and "Zusanli" (ST 36) of right side with disperse-dense wave, 4 Hz/20 Hz in frequency and 0.2 mA in current intensity, 30 min each time, once a day for lasting 7 consecutive days. The reduction rate of cerebral blood flow was measured with laser Doppler flowmetry during operation. The neurological function of rats was observed using Zea Longa neurobehavioral score. The cerebral infarction volume was detected by TTC staining method. The microglia positive expression in the ischemic side of the cortex was detected with the immunofluorescence method. Under transmission electron microscope, the ultrastructure of cell in the ischemic cortex was observed. The mRNA expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1) and gasdermin D (GSDMD) in the ischemic cortex were detected using real-time PCR.@*RESULTS@#Compared with the sham-operation group, in the model group, the reduction rate of cerebral blood flow was increased during operation (P<0.001); Zea Longa neurobehavional score and the percentage of cerebral infarction volume were increased (P<0.001), the numbers of M1-type microglia marked by CD68+ and M2-type microglia marked by TMEM119+ were elevated in the ischemic cortex (P<0.001), the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was increased (P<0.001, P<0.01); the cytomembrane structure was destroyed, with more cell membrane pores formed in the ischemic cortex. Compared with the model group, after intervention, Zea Longa neurobehavioral score and the percentage of cerebral infarction volume were reduced (P<0.05), the number of M1-type microglia marked by CD68+ was reduced (P<0.05) and the number of M2-type microglia marked by TMEM119+ was increased (P<0.05); and the mRNA expression of NLRP3, ASC, Caspase-1 and GSDMD was decreased (P<0.01, P<0.05) in the EA group. Even though the cytomembrane structure was incomplete, there were less membrane pores presented in the ischemic cortex in the EA group after intervention.@*CONCLUSION@#The intervention with EA attenuates the neurological dysfunction and reduces the volume of cerebral infarction in the rats with cerebral ischemic reperfusion. The underlying mechanism is related to the inhibition of microglia pyroptosis through modulating NLRP3/Caspase-1/GSDMD axis.